In this competitive renewal application, our experimental plan remains focused on identifying the primary molecular targets and the key signaling molecules by which the cancer inhibitory activity of energy restriction is exerted. Our objectives are stated in the following specific aims. 1) Can patterns of gene and/or protein expression in the mammary gland identify: those animals that are energy restricted versus those that are ad libitum fed, and those animals that will be protected against mammary carcinogenesis by energy restriction versus those energy restricted animals in which mammary carcinomas will occur? The initial hypothesis that will be tested is that the inhibitory effect can be detected as differential patterns of gene and/or protein expression in the mammary gland of energy restricted versus ad libitum fed rats. This line of inquiry will then be extended to investigate why some animals fail to respond to energy restriction. Current evidence supports the likelihood that detailed analyses will be directed at the investigation of signaling/metabolic pathways of which AMPK and PI-3 kinase are components. Alternative hypotheses would examine regulatory effects in epithelial cells isolated either from the mammary gland or from pre-malignant mammary pathologies as the target of energy restriction. 2) What groups of related genes and/or proteins are differentially expressed in mammary carcinomas that occur in energy restricted animals versus the carcinomas that occur in ad libitum fed animals? We hypothesize that certain molecular alterations in carcinomas will allow them to tolerate the effects of limited glucose availability and permit their emergence despite the imposition of energy restriction. We argue that these effects will be reflected in the energy charge of carcinomas, their levels of AMPK activity, and the activity of the PI3K-Akt signaling pathway. We anticipate that these studies will complement those in Aim 1 and provide a comprehensive understanding of the effects of energy restriction on both the mammary gland and pre-malignant and malignant stages of the carcinogenic process. 3) Can an energy restriction mimetic agent such as 2-deoxyglucose inhibit mammary carcinogenesis in ad libitum fed animals? We predict that it will be possible to use a glucose analogue, e.g. 2-deoxyglucose (2-DG), to differentially impose energy restriction on cells in developing pre-malignant mammary pathologies. Our hypothesis is that 2-DG will block glucose metabolism in pre-malignant mammary pathologies and inhibit their progression to mammary carcinomas without inducing adverse effects on non-transformed cells. The work proposed in this application has the potential to identify the key molecular events by which energy restriction inhibits the carcinogenic process and to provide guidance on the selection of surrogate endpoint biomarkers for clinical intervention studies. Moreover, this work may elucidate 1) why some cancers are not responsive to the inhibitory effects of energy restriction, 2) whether limiting glucose availability is a key to protection against cancer, and 3) a basis for the development of energy restriction mimetic strategies to prevent breast cancer occurrence.